The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD(+)-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD(+)-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

The procedure-associated mortality rate of bone marrow transplantations (BMT) remains close to 25%. The BMT is therefore only indicated for life-threatening diseases, with no replacement of this technology to be expected in the foreseeable future. Modest improvements such as the use of G-CSF to accelerate hematopoietic recovery significantly reduce mortality. Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and are mainly quiescent, dividing only to self-renew. Via extracellular signals and interactions with other cells of the BM (microenvironment), a stable HSC pool is maintained, while hematopoietic progenitor cells (HPC) proliferate and differentiate to mature blood cell types. Marrow adipocytes (BMA) have recently been recognized as a hematopoietic regulatory entity, although the mechanisms by which it interacts with HSCs and HPCs (HSPC) in vivo, remain unknown. During the hematopoietic recovery period post-BMT, HSPCs increase their metabolism as a response to hematopoietic stress, partly modulated by the microenvironment. In this work we identified novel pharmacological approaches to accelerate hematopoietic recovery in the post-BMT period, addressed via two strategies: (i) by directly modulating HSC metabolism, and (ii) by regulating the differentiation of BMA. Increasing oxidative phosphorylation, combined with an impaired mitochondrial stress response, can severely compromise the regenerative capacity of HSCs. Here we demonstrate that the NAD+-boosting agent Nicotinamide Riboside (NR) reduces mitochondrial activity within HSCs through increased mitophagy, the recycling mechanism of damaged mitochondria. We observed in vitro NR treatment of HSCs to lead to increased asymmetric HSC divisions, which resulted in a significantly enlarged pool of progenitor cells, without HSC exhaustion. Dietary supplementation of NR to mice undergoing BMT accelerated blood recovery and improved survival. We therefore established a novel link between HSC mitochondrial stress, mitophagy and stem cell fate decision. Recent studies have suggested BMA maintain HSCs quiescent, which, may be detrimental to the increased expansion of HPCs in the recovery phase post-BMT. BM stromal cells (BMSC) are the cellular precursors of both adipocytes and osteoblasts, and they have been strongly implicated in supporting hematopoiesis by secreting cytokines such as SCF and Cxcl12. We observed that adipocytes quickly accumulate the marrow space of mice undergoing BMT, followed by an expansion hematopoiesis-supportive multipotent Sca1+/CD24+ stromal cells, coinciding with hematopoietic recovery. We developed a novel screening platform using Digital Holographic Microscopy (DHM), quantifying in vitro adipocytic differentiation non-invasively, allowing for follow-up co-cultures with HSPCs. Using the OP9 BMSC-line, we modulated adipocyte differentiation prior to co-culture using a pharmacological approach and observed that high adipocytic content was associated to a decrease in HPC expansion. We perfomed a screening of over 4000 FDA-approved drugs and natural compounds and identified one to efficiently prevent adipocytic differentiation and maintain the hematopoietic supportive capacity of OP9 stroma. Altogether, this work opens the door to alternative strategies accelerating hematopoietic reconstitution and unveils the potential to improve recovery of patients undergoing BMT.

EPFL2019

Novel tools to dissect stem cell metabolism at single cell level

Gennady Nikitin

Hematopoietic stem cells (HSC) are responsible for the life-long maintenance of our blood system. Their long-term capacity to both self-renew and differentiate and the ability to efficiently âhomeâ to their bone marrow niches when injected in the blood stream, makes these rare cells ideal candidates for transplantation for the treatment of various blood cancers. However, countless attempts to expand HSC in vitro without losing their stem cell potential have failed, which is, to a large extent, linked to our poor understanding of the mechanisms that control HSC fate. Recent discoveries have revealed a crucial role of metabolism in controlling HSC function in vivo. However, because of major technical difficulties, we do not yet know the underlying mechanisms behind the metabolic control of HSC fate. Traditional, population-level experimental approaches link the fate of a stem cell to a cell surface protein expression phenotype. This method does not take into account the large heterogeneity of metabolic states in HSC. Consequently, to get mechanistic insights on HSC fate decision-making, metabolism must be studied at single cell level. Therefore, the overall goal of this thesis is to establish novel tools to study metabolic regulation of HSC at single cell level. Here we specifically focused on two functional metabolic read-outs, protein turnover and mitochondrial pH. Mitochondrial matrix pH is one of the most important functional parameters of mitochondria, as it is directly related to the efficiency of ATP production and thus reflects the metabolic state of the mitochondria. Here, a new ratiometric fluorescent probe, 5FA-PIP-Cy5-DA, able to reliably quantify mitochondrial pH, has been developed. It selectively accumulates in mitochondria of live cells and can be used to sensitively detect mitochondrial pH fluctuations upon various external stimuli. Spectral properties of 5FA-PIP-Cy5-DA allow it to be combined with the total mitochondrial membrane potential-dependent probes, enabling simultaneous imaging of total mitochondrial potential and mitochondrial pH. To complement the data on mitochondrial pH in live cells with an additional independent parameter of mitochondrial metabolism, a method of correlated TEM and nanoscale secondary ion mass-spectrometry (NanoSIMS) imaging was developed, that for the first time allows to study the subcellular protein turnover in single stem cells. Application of these novel tools to study HSC metabolism revealed a striking correlation between the levels of mitochondrial protein turnover and mitochondrial pH. This correlation reflects the burst in mitochondrial metabolism upon HSC activation. Intriguingly, an extreme burst in both mitochondrial pH alkalinization and protein turnover rate was observed for the most potent long-term HSC (LT-HSC) upon their activation and induction of differentiation. A new model is proposed to explain the link between the observed activation of mitochondrial protein structure reorganization and increase in mitochondrial pH level of LT-HSC undergoing differentiation. Finally, to demonstrate the broad range of applicability of the new mitochondrial pH probe, we applied it to measure the impact of anticancer treatment on mitochondrial metabolism in ovarian cancer cells exposed to two anticancer drugs, RAPTA-T and cis-platin. Distinctly different mitochondrial pH responses were measured for these two drugs, revealing alternative mechanisms behind their impact on mitochondria.

EPFL2018

Metabolic rescue of muscle and muscle stem cells in muscle disease

Peiling Luan

Duchenne muscular dystrophy (DMD), caused by the mutation of dystrophin gene, is an X-linked disorder that affects 1 in 3500 males, leading to progressive muscle degeneration and eventually resulting in premature death. Increasing evidence indicates that DMD and age-related sarcopenia share clinical hallmarks, such as decreased metabolic activity of both myofibers and muscle stem cells (MuSCs), which results in defective MuSC function and impaired muscle regeneration. In my Ph.D. thesis, I focused on manipulating mitochondrial function, such as mitophagy, and lipid metabolism, particularly sphingolipid metabolic pathways, to restore muscle and MuSC functions in DMD and age-associated sarcopenia. My thesis consists of three projects: Rescue of mitophagy improves muscle function in DMD. In this project, we identified defective mitophagy as a hallmark of muscular dystrophy. Rescue of dysfunctional mitophagy by dietary supplementation of Urolithin A (UA), a metabolite present in fruit extracts and a potent activator of mitophagy, improved muscle function in mdx mice. The beneficial effects of mitophagy restoration observed in dystrophic animals were attributed to the improvement in mitochondrial function, structural integrity of muscles, restoration of MuSC activity, and reduction in general inflammation and fibrosis. Sphingolipid depletion improves muscle regeneration during ageing. We first established the link between sphingolipid metabolism and ageing by demonstrating that sphingolipids accumulate in skeletal muscle upon aging. We reduced skeletal muscle sphingolipid accumulation by treating aged mice with myriocin, a selective inhibitor of serine pamitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid de novo synthesis pathway. Sphingolipid depletion increased MuSC regenerative ability through enhancing the proliferation and differentiation capacities of MuSCs, ultimately leading to the prevention of age-related decline in muscle mass and function. Inhibition of sphingolipid synthesis reverts muscular dystrophy. By studying skeletal muscle transcript profiles of human muscular dystrophies, we identified upregulation of sphingolipid biosynthetic pathway as a universal feature of human patients affected with muscular dystrophy. To rescue the aberrant sphingolipid metabolism, we treated mdx mice with myriocin and showed that inhibition of sphingolipid generation enhanced muscle function, leading to amelioration of DMD. Furthermore, we uncovered a novel role of sphingolipid depletion in macrophage polarization and in reconstruction of the mdx MuSC niche, resulting in improved regenerative capacity of MuSCs. In summary, our work establishes the essential roles of mitophagy and sphingolipid metabolism in maintaining muscle and MuSC function. Restoration of mitophagy and blockade of sphingolipid generation could therefore be attractive treatment strategies to delay the progression of DMD and age-related sarcopenia.

EPFL2020